The invention relates to cultivation of cells, and more particularly to tissue propagation and product retrieval under axenic conditions.
The cultivation of cells for metabolic and other products is rapidly gaining in importance, as the laboratory production of these products becomes economically infeasible, or is technologically impossible. Moreover, recombinant DNA techniques and related technologies has made possible the generation of cell lines which can produce more numerous products in heretofore unknown quantities. Accordingly, cultivation of cells and the attainment of high yields is a primary objective for an increasing number of manufacturing enterprises.
Conventional plant tissue culture methods rely on the provision of minerals, growth regulators and a carbon source, usually sucrose, to plant tissue by incorporating these ingredients in an agar solidified gel. Plant tissue is placed on the surface of the gel and nutrients are taken up from the gel. This method of culture has several inherent problems, all of which mitigate against the optimal growth of the plant tissue. Many initial explants and some callus cultures produce and introduce into the medium, compounds which inhibit the growth of the plant tissues by either slowing the cell division rate, or by killing the tissue. Such diffusion necessitates the frequent transfer of the tissue to fresh media, involving both the use of extra medium and its preparation, as well as labor. Since the plant tissue is necessarily growing in close contact with the medium, the maximum availability of oxygen is limited to the upper side of the callus or tissue. This restricts the respiration rates of the lower side of the callus and growth in that region is often depressed. Plant tissue, both differentiated and undifferentiated, produce volatile growth regulators, notably ethylene, which affects the form and growth rate of the tissue. In conventional culture vessels, frequently petri dishes or other plastic or glass containers, these volatile compounds can build up and prejudice the efficient growth of the tissue. Because all the nutrients, growth regulators and carbon sources for the growth of the plant tissue must diffuse through the gelled medium, the growth rate of the tissue is limited by the rate of this diffusion.
Suspension cell culture has been used to alleviate some of the problems in plant culture, and is widely used in the cultivation of many cell types. In plant cell culture, suspension cultures are induced by manipulations of the growth regulator component of the medium to produce embryos. The cultures require regular subculturing and can suffer from depletion of the nutrients or build-up of noxious compounds in the medium if not transferred regularly. Aeration in these cultures is usually provided by agitation of the culture vessel; thus, little control is exercised over water soluble volatile gases such as ethylene. Aeration remains a predominant problem in the culture of all cell types by suspension methods.
Other cultivation methods include multiple plate suspension propagators, glass bead propagators, and tubular spiral films. Multiple plate propagators comprise a number of support layers within a liquid suspension. This system suffers from the same disadvantages as other suspension cultivators, particularly, poor gas diffusion and the accumulation of harmful products within the suspension. Glass bead propagators comprise glass beads coated with cells. The beads provide an increased surface area for cell attachment. Not only does this method fail to solve gas diffusion limitations, but subjects the cells to great mechanical stress, resulting in high cell loss and concomitant low yields. In the use of tubular spiral films or hollow permeable fibers, cells are introduced to the inside of a tube which is permeable to gases. Liquid nutrient is flowed through the inside of the tube. This approach also suffers from diffusion problems, in that cells disposed inward from the lining cells are exposed to increasingly lower amounts of diffused gases.
Accordingly, it is an object of the invention to provide a method and apparatus for the cultivation of cells and tissues. It is a related object to provide for the cultivation of one cell type in axenic conditions.
It is a further object to provide for heretofore unrealized high gas availability to the cultivated cells, as well as the unlimited supply of solubilized nutrients.
It is an additional object to provide for the improved growth of most cell types, including plant cells and hybridomas, by a single apparatus design.
It is yet another object to provide for reduced labor requirements in the cultivation of plant and other tissue types. Further, it is an object to provide highly consistent, repeatable cell cultivation by efficient, low cost apparatus and processes. A related objective is the provision of a cultivating system which is self-adjusting and microprocessor controllable, whereby a minimum amount of human interaction is required for generating high product yields.